The long-term goal of this work is to understand the neuroendocrine mechanisms involved in the regulation of PRL secretion. The existence of a putative novel PRF was discovered by the applicant. PRF is a small peptide found in the intermediate lobe of the pituitary. PRF is effective both in vitro and in vivo and apparently is critical for the regulation of PRL release during lactation, in the ovulatory cycle and in other conditions. The main thrust of the proposed studies will be isolation, sequencing and synthesis of ovine PP PRF. Sequential purification on HPLC columns, mass spectrometry, amino acid analysis and microsequencing will be utilized. PRF will be synthesized by a solid phase method and its biological activity validated in vitro and in vivo. Polyclonal antibodies to synthetic PRF will be generated. The distribution, function and regulation of PRF will be determined. Radioimmunoassays, ELISAs and immunocytochemical methods for studying PRF will be developed. The distribution of PRF within the hypothalamo-hypophysial system and other sites will be determined. The physiological functions of PRF will be determined using passive immunization. The factors affecting PRF release from cultured PP cells will be determined. Interactions between anterior pituitary (AP) and PP cells will be examined by co-culturing PP cells and primary AP cells or GH3 cells and examining PRL synthesis. The proliferation of lactotrophs will be examined and the subpopulation of PP cells involved will be identified.